Albumin is found in a variety of biological samples, including plasma, amniotic fluid, seminal fluid, and cerebral spinal fluids, among others. Albumin is highly abundant in such samples, and in particular, constitutes more than 50% of total plasma proteins. Often, however, it is desirable to identify low abundance or low molecular weight proteins or peptides present in the sample, since such proteins or peptides may serve as important biomarkers for a variety of diseases, including cancer. Two-dimensional (2D) polyacrylamide gel electrophoresis is the preferred platform of large-scale protein microcharacterization, as it allows for qualitative and quantitative analysis of the proteome. However, the separation of low abundance proteins from plasma or serum samples by 2D gel is complicated by the high abundance of albumin present in human serum. In many cases this arises from limitations in protein loading on the isoelectric focusing gel (first dimension of the separation) using an Immobilized pH Gradient (IPG) strip. Therefore, by significantly depleting the amount of albumin present in a plasma sample, it is expected that visualization of the other plasma proteins will be improved, and increased total protein load will enable the detection of less abundant proteins.
In addition, albumin is known to bind to a variety of substances present in serum or plasma samples, including various peptides, proteins, protein fragments, hormones, cytokines, fatty acids, and other small molecular biomarkers that may contain important diagnostic information. Since albumin may bind to these biomarkers, these biomarkers are often eliminated from the biological sample along with the albumin by traditional separation and fractionation techniques. It would therefore be advantageous to be able to separate albumin from a biological sample, without simultaneously eliminating biomarkers that may be bound to the albumin.
Several techniques have been used for the elimination or reduction of albumin from serum samples. One such technique uses Cibacron blue dye. One example of a Cibacron blue-related method uses a crosslinked agarose bead with covalently attached Cibacron blue F3GA dye (Affi-Gel Blue, Bio-Rad, Hercules, Calif.). The attached dye functions as an ionic, hydrophobic, aromatic, and sterically active binding site for proteins. Other reports indicate that Cibacron Blue F3GA dye has a specific affinity to most proteins that contain a dinucleotide fold, therefore mimicking nicotinamide adenine dinucleotide and other purine dinucleotides. However, given the multiple functions of the attached dye, the total removal of albumin using the Affi-Gel Blue method will not only clear albumin and many proteins with the same isoelectric point (pl), but also the proteins that can bind to the planar ring structure of Cibacron Blue F3GA dye through a complex combination of electrostatic, hydrophobic and hydrogen-bonding interactions. For example, biomolecules such as interferon, lipoproteins, hemopexin, antithrombin II, blood coagulation factors, nicotinamide adenine dinucleotide, and other purin dinucleotides may also be bound. Thus, because of the multiple dye interactions and binding of non-albumin proteins and other molecules, methods based on the use of the Cibacron blue dye only provide a partial separation of albumin from other proteins.
Isoelectric trapping and Gradiflow™ separation have also been used for the elimination of albumin from a sample. Isoelectric trapping uses multicompartment electrolyzers with isoelectric membranes. This device acts to purify proteins to homogeneity in a liquid vein by capturing them in an isoelectric trap formed by two immobiline membranes with pls encompassing the pI of the species under analysis. Gradiflow™ separation is capable of separating proteins on the basis of their molecular weight and charge by selection of a specific separation membrane cutoff size and adjusting the pH of the system. These techniques, however, have several disadvantages including the high cost of the instrumentation and the frequent maintenance, the lengthy treatment time of the serum sample, and the loss of several plasma proteins during processing, including proteins with a pI the same as or close to that of albumin. In addition, the conditions under which isoelectric trapping is performed may result in the denaturing of proteins in the sample, rendering these proteins unsuitable for further examination.
Other techniques have utilized antibodies which bind albumin as the albumin containing sample passes through a column. Although such techniques provide for relatively good separation of albumin from other proteins present in the sample, some cross reactivity with other proteins may occur. In addition, the antibodies used in such techniques are not always stable (i.e., they may be denatured by running conditions or digested by proteinases present in plasma samples), and the cost may be prohibitive.